bioRxiv preprint doi: https://doi.org/10.1101/864678; this version posted December 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 bioRxiv preprint doi: https://doi.org/10.1101/864678; this version posted December 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami Emanuela Torelli1,*, Jerzy W. Kozyra1,§, Ben Shirt-Ediss1, Luca Piantanida2,^, Kislon Voïtchovsky2, and Natalio Krasnogor1,* 1Interdisciplinary Computing and Complex BioSystems (ICOS), Centre for Synthetic Biology and Bioeconomy (CSBB), Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RX, United Kingdom 2Department of Physics, Durham University, Durham, DH1 3LE, United Kingdom §Present Address: Nanovery Ltd, 85 Great Portland Street, First Floor, London, W1W 7LT, United Kingdom ^Present Address: Micron School of Materials Science & Engineering, Boise State University, Boise, ID 83725, USA *[email protected], [email protected] 2 bioRxiv preprint doi: https://doi.org/10.1101/864678; this version posted December 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT The scaffolded origami technique has provided an attractive tool for engineering nucleic acid nanostructures. This paper demonstrates scaffolded RNA origami folding in vitro in which all components are transcribed simultaneously in a single-pot reaction. Double-stranded DNA sequences are transcribed by T7 RNA polymerase into scaffold and staple strands able to correctly fold in high yield into the nanoribbon. Synthesis is successfully confirmed by atomic force microscopy and the unpurified transcription reaction mixture is analyzed by an in gel-imaging assay where the transcribed RNA nanoribbons are able to capture the specific dye through the reconstituted split Broccoli aptamer showing a clear green fluorescent band. Finally, we simulate the RNA origami in silico using the nucleotide-level coarse-grained model oxRNA to investigate the thermodynamic stability of the assembled nanostructure in isothermal conditions over a period of time. Our work suggests that the scaffolded origami technique is a valid, and potentially more powerful, assembly alternative to the single-stranded origami technique for future in vivo applications. KEYWORDS Co-transcriptional folding, scaffolded RNA origami, bio-orthogonal, split Broccoli aptamer, oxRNA simulation 1 Introduction RNA plays sophisticated roles with different and essential coding and noncoding functions. mRNAs, tRNAs, rybozymes, aptamers and CRISPR RNAs are just few examples of RNA species in a vast functional repertoire. Considering the diversity in functional and structural motifs, the emerging field of RNA nanotechnology has been developing rapidly over the past years. As a consequence, a variety of RNA nanostructures with different functionalities, sizes and shapes have 3 bioRxiv preprint doi: https://doi.org/10.1101/864678; this version posted December 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. been created to investigate and successfully demonstrate their potential in nanobiomedicine and synthetic biology [1-12]. Different self-assembly strategies have been adopted to design RNA nanostructures [13] ranging from RNA architectonics [14-18] to single-stranded self-assembly [19]. Furthermore, taking advantage of the sequential transcription reaction by bacteriophage RNA polymerase, single-stranded RNA origami has been synthesized from long ssRNA molecules. In vitro transcribed and purified RNA sequences were self-folded into hearts, rectangles and rhombus shapes adapting the ssDNA origami design strategy and considering the helical periodicity difference between DNA B-type and RNA A-type helix. Large and complex ssRNA origami were synthesized using partially complemented double-stranded RNA and parallel cross-over cohesion without limitation due to RNA kissing loop interactions [20]. However, these multikilobase ssRNA nanostructures were folded using a thermal annealing ramp gradient (from 85 °C to 25 °C), thereby limiting potential in vivo applications, such as the scaffolding of enzymes [21]. On the other hand, previous work showed the synthesis of smaller ssRNA origami tiles and hexagonal lattices made by annealing and/or in vitro co-transcriptional folding that should be compatible with in vivo folding when genetically encoded and expressed in cells [22]. These authors developed a strategy based on the combination of hairpins, kissing loops and “dovetail seam” to promote and stabilize the folding during the T7 RNA polymerase in vitro transcription reaction [22, 23]. More recently, the ssRNA 2H-AE-ST tile scaffold presented by Geary and coll. [22] was used to create an aptamer-based FRET system where RNA tile synthesis in vivo was demonstrated by measuring FRET outputs without a direct atomic force microscopy (AFM) visualization, due to the small construct dimension [24]. Li and coll. [25] developed a different strategy in which the design concept was similar to the approach reported above, but avoiding the use of short “dovetail seams”. The RNA nanostructures were designed based on natural motifs: the folding pathway was based on hairpin formation and 4 bioRxiv preprint doi: https://doi.org/10.1101/864678; this version posted December 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. tertiary interactions of unpaired residues. In detail, an RNA double-square was designed using 3- way loop observed in phi29 pRNA and 90°-kink from hepatitis C virus RNA genome. These thermodynamically stable and kinetically favourable RNA nanostructures were folded both in vitro and in vivo: nonetheless, the combination of phage and viral derived structural motifs can limit the structure design, the attachment of functional units, the creation of reconfigurable and dynamic nanostructures or their suitability for future theranostic in vivo applications. In addition to architectonics, single-stranded self-assembly and single-stranded origami strategies, the scaffolded RNA origami approach is still in its infancy. Drawing from DNA origami techniques in which several short staple strands sequences promote the folding of a longer single stranded scaffold into a specific shaped structure [26], we designed and synthesised RNA origami following a similar strategy. This approach can provide several advantages such as the high synthesis yield, and the possibilities to produce reconfigurable nanostructures and to incorporate multiple and different functionalities in a precise position. Indeed, previous works demonstrated the synthesis of chemically modified and siRNAs functionalized RNA origamis following a thermal gradient annealing of RNA staple strands and scaffold [27-28]. In our previous work, we went a step further to develop a biologically afunctional (i.e. bio- orthogonal by design) RNA origami able to fold at constant temperature (37 °C) after an initial denaturation step [29], which lights-up when folding to its target configuration. Seven RNA staple strands promoted the folding of a RNA bio-orthogonal synthetic De Bruijn scaffold sequence (DBS) that does not contain genetic information, restriction enzyme sites or reduced ambiguity in the addressability [30]. We demonstrated the possibility to combine scaffold bio-orthogonality, physiologically compatible folding and assembly monitoring using a new RNA-based reporter system. The folding was monitored using our new split Broccoli aptamer system [29]. Motivated by our previous study, and with an ultimate goal of enabling RNA origami expression in living cells, here we demonstrate a full isothermal protocol for scaffolded RNA origami assembly 5 bioRxiv preprint doi: https://doi.org/10.1101/864678; this version posted December 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. via co-transcriptional folding. We maintain the design simplicity and the scaffold nucleotide composition, while changing each staple in order to guarantee a reasonable yield of the desired transcripts and a low aberrant products synthesis during the in vitro transcription by T7 RNA polymerase. The RNA origami is co-transcriptionally folded into a nanoribbon shape at 37 °C: in detail, during the scaffolded RNA assembly, scaffold and staple strands are in vitro transcribed and folded in a one-pot reaction. The RNA origami assembly is verified by gel assay and well characterized by AFM. The RNA nanostructures self-assembly is also successfully confirmed and selectively detected using our split Broccoli aptamer system: the transcription mix is analyzed by in-gel imaging and the tagged RNA origami shows a clear fluorescent band. Finally, the assembled RNA origami nanoribbon is visualized and simulated at equilibrium using the oxRNA coarse- grained model. The in vitro transcribed and folded RNA origami described